Secondary structure and orientation of the pore-forming toxin lysenin in a sphingomyelin-containing membrane.

Lysenin is a sphingomyelin-recognizing toxin which forms stable oligomers upon membrane binding and causes cell lysis. To get insight into the mechanism of the transition of lysenin from a soluble to a membrane-bound form, surface activity of the protein and its binding to lipid membranes were studied using tensiometric measurements, Fourier-transform infrared spectroscopy (FTIR) and FTIR-linear dichroism. The results showed cooperative adsorption of recombinant lysenin-His at the argon-water interface from the water subphase which suggested self-association of lysenin-His in solution. An assembly of premature oligomers by lysenin-His in solution was confirmed by blue native gel electrophoresis. When a monolayer composed of sphingomyelin and cholesterol was present at the interface, the rate of insertion of lysenin-His into the monolayer was considerably enhanced. Analysis of FTIR spectra of soluble lysenin-His demonstrated that the protein contained 27% beta-sheet, 28% aggregated beta-strands, 10% alpha-helix, 23% turns and loops and 12% different kinds of aggregated forms. In membrane-bound lysenin-His the total content of alpha-helices, turns and loops, and beta-structures did not change, however, the 1636cm(-1) beta-sheet band increased from 18% to 31% at the expense of the 1680cm(-1) beta-sheet structure. Spectral analysis of the amide I band showed that the alpha-helical component was oriented with at 41 degrees to the normal to the membrane, indicating that this protein segment could be anchored in the hydrophobic core of the membrane.